Yersinia pestis, the etiologic agent of plague, uses a type III secretion system (TTSS) to translocate virulence proteins, termed Yops, into eukaryotic cells. Yop export is triggered by contact with a host cell in vivo or by growth in the absence of Ca2+ in vitro. The Y. pestis YopN, TyeA, SycN, and YscB proteins are required to prevent Yop secretion in the presence of Ca2+ and prior to contact with a eukaryotic cell. Previous studies have established that the SycN and YscB proteins function as specific chaperones for the secreted YopN protein. A complex of SycN and YscB, but not SycN or YscB alone, directly binds to an N-terminal domain of YopN. TyeA, on the other hand, binds to a C-terminal domain of YopN. This project will analyze the function of the YopN-TyeA-SycN-YscB complex in the regulation of Yop secretion. The control of virulence protein secretion is of broad interest because of the realization that numerous bacterial pathogens employ such "cell contact-dependent" delivery systems. The goals of this project are: (i) to define the function of the SycN/YscB chaperone complex in YopN secretion, YopN translocation and in the regulation of Yop secretion; (ii) to identify the mechanism by which the YopN-TyeA-SycN-YscB complex blocks secretion; and (iii) to identify the signal, sensors and signal transduction pathways that lead to the removal of the YopN-TyeA-SycN-YscB-dependent block in Yop secretion. The objectives of this project will be realized through the selection and analysis of specific Y pestis mutants that fail to block Yop secretion, constitutively block Yop secretion, or effect specific protein-protein interactions within the YopN-TyeA-SycN-YscB complex. Immunoprecipitation experiments and yeast two- and three-hybrid analyses will be used to identify and characterize novel protein-protein interactions. Biochemical and biophysical analyses will be employed to determine the stoichiometry, conformation and binding constants of specific proteins or protein complexes. A Y. pestis chromosomal transposome insertion library will be screened to identify specific insertion mutants that are defective in Yop secretion or in the regulation of Yop secretion. These studies will provide insight into the role of the YopNTyeA-SycN-YscB complex in the regulation of Yop secretion in Y. pestis.